This invention relates to an improved probe which detects the presence of biological material and, more particularly, a probe designed to detect the presence of preselected DNA or RNA segments which are characteristic of a particular biological agent at very low levels.
The ability to remotely and automatically detect the presence of specific molecular structures, and particularly the presence of a biologically active structures, is desirable in connection with a wide variety of applications. In environmental and battlefield applications, it is desirable to provide probes which can rapidly detect the presence of highly virulent bacteria, viruses or other organisms which pose a threat to human health. Remote detection of the presence of characteristic deoxyribonucleic acid (DNA) of an organism or virus would enable a highly accurate identification of a biological agent without entering the immediate environment harboring the substance. In view of the potential for the agent to be widely dispersed in air or water, it is also desirable to be able to detect the presence of potential pathogens at very low levels. The detection of pathogens at very low levels can provide an early warning so that responsive protective measures may be quickly implemented. For example, the remote detection of an environmentally released cloud of a biological agent is of vital importance to the military.
A further application where the rapid and accurate detection of low levels of DNA is desirable is in connection with the diagnosis of disease. Biologically active agents, including bacteria and viruses which may cause disease, can be identified by their unique DNA sequences. The rapid and accurate identification of a pathological biological agent from a culture, biopsy or blood sample can assist in the diagnosis of disease and accordingly facilitate an appropriate treatment of a patient by health care professionals at the earliest opportunity. The rapid and accurate detection of the presence of particular segments of DNA or RNA (ribonucleic acid) segments also has applications in connection with genetic screening and in connection with new drug development. For example, probes may be employed for a rapid determination of viral titer in samples after being treated with antiviral or anti-bacterial drugs under investigation to assess their efficacy.
Conventional techniques for identification of unknown biological agents which are frequently invoked include assays which employ antigen-antibody reactions. Although the antibody-antigen assays provide a rapid response, they are not particularly accurate, because often antibodies are not highly specific will bind with more than one antigen. Furthermore, all such assays are not particularly sensitive when only very low levels of the antigen are present. Sensitivity is an assay""s ability to make a detection at very low levels of sample material. In some circumstances it is feasible to culture samples containing unknown organisms to increase the volume available for subsequent inspection and testing by trained laboratory technicians, however these procedures are time consuming, expensive and often not practical. Other traditional assay techniques involve the basic physical measurements of the molecular structures of an unknown sample by optical techniques, mass spectroscopy or nuclear magnetic resonance. These physics-based detection techniques attempt to identify the unknown material by pattern matching against the large databases of known material parameters that have been accumulated over the years. Although these techniques can provide a rapid response, these chemical and biological sensors are often large (e.g., table-top to room-sized), are not highly sensitive or accurate, and require expensive laboratory equipment.
One problem with employing these conventional detection technologies is that they require relatively large volumes of the unknown agent, and due to collection techniques, or because the concentration in the sample of a target organism or virus is very low, the identification methods are not sensitive enough to provide for an accurate positive identification. For example, the concentration of the target biological agent in a sample may be very small in view of a wide dispersal and consequent dilution of the agent in air or water. A further problem and concern with conventional detection technologies is the time necessary to make a positive and accurate identification. The ability to make a real time identification of highly virulent agents would enable the implementation of an immediate and appropriate response.
The advent of polymerase chain reaction or PCR technology has significantly advanced many of the problems associated with the problems with very small sample sizes. The PCR process provides a relative rapid and accurate manner to amplify any DNA within a sample and the process has been incorporated into automated systems for the amplification and detection of nucleic acid sequences for infectious agents. Development efforts are presently underway which combine the PCR process and miniaturization of mechanical components such as putting an electrophoretic, optical or mass spectrometer detector-on-a-chip along with fabricating the adjunct microfluidic components, such as tiny valves and pumps, which are necessary to configured an integrated DNA amplifier and detector. Other related amplification techniques include ligase chain reaction, strand displacement amplification, transcription-associated amplification and nucleic sequence-based amplifications.
Although PCR technology addresses some of the problems with insufficient or inadequate sample size and thereby can improve sensitivity, the integration of these process typically increases the time necessary to complete the identification process. Use of the PCR process essentially forecloses the ability to make detections in real time because it requires successive amplification steps. Further, although PCR provides a solution for problems associated with low sample volumes, the technology does not directly address the identification or detection step. Utilization of PCR technology can reduce the time and increase the accuracy compared with conventional assay techniques however the protocols remain complex, there are contamination problems and the process is time consuming and costly.
A number of gene detection techniques involve the hybridization of target DNA with a complementary nucleic acids sequence on a probe. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of complementary probe molecules. Identification techniques which employ such hybridization steps generally have a high degree of accuracy and can provide a rapid identification of the biological agent. Although these hybridization techniques are highly specific, the low sensitivities of the probes remain a problem associated with these methods. For example, one approach using hybridization of complimentary nucleic acid sequences deals with the sensitivity problem employs PCR-based methodology to amplify the target nucleic acid sequence. The PCR process is followed by a biotinylation step. The amplified products are then captured on a substrate consisting of oligonucleotide-coated paramagnetic microparticles. The detection step employs an avidin-horseradish peroxidase conjugate. Hybridized molecules can be detected by binding to an avidin enzyme complex resulting in a colored product which indicates the presence and/or position of the biotinylated probe.
A further detection technique which employs the hybridization of nucleic acids involves the attachment of fluorescent or radioactive markers on DNA molecules. Hybridization proceeds with oligonucleotides attached to a chip where the locations of particular oligonucleotides have been previously identified. Any DNA in die hybridization mixture flows over the probe and forms a bond to any sites that mimic the opposite strand from which they have a complementary sequence. In one example, when die bonding step is completed, a technician moves the chip into an automated reader where a laser scans the slide row by row, exciting the fluorescent molecules. A computer can then record the pattern of bright and dim blocks, indicating which probes found matching DNA in the test sample. By comparing the pattern to a map of known probe locations, this system can identify unknown genetic sequences.
A disadvantage to the use of markers in the detection step is that the radioactive materials and flourescent dyes have mutagentic properties. The synthesis of the probes requires a step attaching the markers to the oligonucleotides which exposes the technician to potentially hazardous substances. Besides the handling dangers in the synthesis of the probes, the ultimate disposal of the assay also remains a concern. These techniques also suffer from sensitivity problems particularly when the target DNA in the sample is very low.
A further biological agent sensing technology which employs hydbridization of DNA or RNA molecules involves the use of an array of microcantilever beams on a chip to which biological materials are selectively attracted. The attachment of the agent to a specific microcantilever can then be detected through measurable changes in its mechanical behavior, specifically, its vibration resonance frequency. Larger matrix arrays of cantilevers can be fabricated and individual beams selectively coated with oligonucleotides by applying electrical voltages only to the selected beam or beams while flooding the chip with the oligonucleotides. Washing and flooding with another oligonucleotide, and a different pattern of applied voltages, can be repeated until the whole array is loaded. When the chip is exposed to an unknown (but anticipated) nuclear material, hybridization will take place only at the locations prepared specifically for that material, which can then be determined by surveying the mechanical properties of the individual locations in the array. Other approaches using hybridization include surface plasma resonance (See Peterlinz, K. A., Georgiadis, R. M. Herne, T. M. and Tarvol, M. J. (1997) Observation of hybridization and Dehybridization of Thiol-Thethered DNA Using Two-Color Surface Plasma Resonance Spectroscopy. J. Am. Chem. Soc. 119, 3401-3402) or quartz crystal microbalance (See Okahata, Y. Matsunobo, Y., Ijiro, K. Mukae, M. Murakmi, A., and Makino, K. (1992), Hybridizatioin of Nucleic Acids Immobilized on a Quartz crystal Microbalance, J. Am Chem Soc. 114, 8299-8300). These publications are hereby incorporated by reference.
As disclosed by Meade et al. in U.S. Pat. No. 5,591,578 entitled Nucleic acid Mediated Electron Transfer, oligonucleotides may be modified with electron donor and acceptor moieties prior to attachment to an electrode. As previously discussed, an oligonucleotide having a nucleotide sequence complementary to a target nucleotide sequence in the DNA of an organism will hybridize with its complementary region located within the DNA strand. The technique disclosed by Meade, which results in a covalent attachment of the moiety to the ribose backbone of a DNA or RNA molecule, only minimally interferes with the molecular structure and the modified molecules retain their characteristic base pairing ability. Megerle disclosed a biological agent probe in U.S. Pat. No. 5,874,046 which can be constructed using this technology which involves the attachment of a plurality of these modified oligonucleotides to an electrode. The Megerle patent is hereby incorporated by reference. In the event target DNA is brought into contact with the probe, multiple hybrids can form on a single electrode, one for each oligonucleotide bound to the electrode under ideal conditions. The probe disclosed by Megerle takes advantage of the changes in the electrical properties of the modified hybridized molecules which results from the base pair bonding. When voltage is applied to the electrode, current will flow through the modified hybridized molecules with little resistance. Measurement of the current, or changes in the current within the sample cell can indicate the presence of target DNA.
Although this technique is both highly accurate and rapid, it nevertheless requires a sufficient sample available in the hybridization mixture to make detection measurements and, when the number of hybridized pairs of the modified molecules is very low, the presence of the target DNA is difficult to detect. When the number of hybridized pairs is very low, electrical current flowing between the electrode and through the electron donor or acceptor group may be to low for the electrical circuitry of the detection device to detect. In addition to the sensitivity problems which are inherent when only small samples are available to test, the sensitivity of probes using hybridization techniques are further adversely affected by steric hindrance of the target nucleotide molecules. In this regard, when the relatively long molecules of denatured single stranded DNA bind with the oligonucleotides attached to the electrode, segments of the molecule on the free ends which did not bind may physically interfere with the ability of additional molecules to hybridize with neighboring oligonucleotides.
Accordingly, it is an object of the invention to provide a highly accurate and highly sensitive probe which can make a rapid detection of the presence of target DNA or RNA. A further object of the invention is to provide to an accurate and sensitive probe which can rapidly detect the presence of potential pathogens A further object of the invention is to provide a biosensor device which can automatically and remotely sample the environment and communicate an output relating the presence or absence of a particular target DNA or RNA. Yet a further object of the invention is to provide an improved sensor cell for a biological agent probe with heightened sensitivity.
The present invention addresses some of the problems of the prior art and more particularly, is directed at improving the sensitivity of the biological probes such as those disclosed in the patent to Megerle U.S. Pat. No. 5,874,046 (hereinafter the Megerle Patent). As disclosed in the Megerle Patent, real time, or close to real time detection of microorganisms can be achieved by employing modified, single-stranded oligonucleotide sequences within a probe which are complementary to characteristic segments in target DNA or RNA. The modification of oligonucleotides by the attachment of electron donor and acceptor moieties can significantly alter the electrochemical properties of the molecule upon hybridization. As disclosed by Meade et al., upon hybridization of the probe DNA which have been modified and target DNA, the conductivity of the hybridized molecule dramatically increases and the conductivity may be detected by electrochemical measuring techniques. While Megerle previously taught the attachment of a plurality of identical oligonucleotides sequences to a single electrode employing the modification technique disclosed by Meade et al., the present invention involves attachment of a plurality of different oligonucleotide groups, each group having a multiple members with identical nucleotide sequences, to a single electrode. Providing a plurality of unique complementary oligonucleotide groups to a single electrode increases the sensitivity of the probe by both diminishing the steric hindrance effects on the surface of the probe such as crowding and tangling and by providing more potential bonding sites per molecule of the target DNA. These characteristics result in a probe having increased sensitivity and which is particularly effective for the detection of DNA or RNA of target biological agents at very low levels. A method of construction of probes having multiple groups of oligonucleotide on a single electrode groups and the operation of such probes in connection with the detection of different target biological agents is also disclosed. The invention provides both a highly accurate identification technique which is sensitive at very low levels and, by employing a hybrid pair which is capable of mediated electron transfer, provides rapid results.